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FLYRM LAB PROTOCOL
Immunocytochemistry Protocol for adult nematodes
September 2010 (developed by K. Susman)
1. Prepare three 6cm plates for each genotype so that they are day 5 on the first day of this procedure (just past food, but not starved). Collect worms into 1.5 ml microfuge tubes by washing off plates with 1 ml M9. Spin at 6000 rpm for 30 sec. Remove most of supernatant.
2. Wash 2 times with 1 ml d H2O. Spin and remove as before.
3. Remove most of H2O, add 500 µl ice-cold 2% Paraformaldehyde in 1X PBS. This solution should be made fresh, approx. the day before use. Store in fridge. Place bottle on ice before beginning procedure.
Heat 50 ml 1X PBS on hot plate to about 55 °C. Dissolve 1 g using a stir bar. Add a few drops of 10 N NaOH. Filter with #4 Whatman paper and funnel into glass bottle. Store in fridge. Be careful not to breathe the fumes.
4. Prepare a dry ice/ethanol bath in a plastic beaker, with a stir bar on stir plate. When this is at –20°C, freeze the tubes (using circular tube holder) for about 1 min. Then, thaw rapidly using 80°C heat block. REPEAT for a total of 3 x.
5. Rock the tubes in cold room for four hours.
6. Spin down worms at 6000 rpm for 30 sec, remove most of supernatant. Add 500 µl 1X PBS to rinse- suspend, spin down and remove supernatant.
7. Add 500 µl BME, 1% triton-x-100, 120 mM tris pH 7 and incubate on rocker in 37°C incubator overnight. [Make this solution up in advance]
Make 10 ml: 100 µl triton-x-100; 500 µl ß-mercaptoethanol, 1.2 ml Tris buffer pH 7.0. TO 10 ml with dH2O. Make this in the fume hood!!
8. Spin down worms and remove supernatant. Add 500 µl 1X PBST (make in advance):
1X PBS; add 50 µl triton-x-100 in a 10 ml solution in a falcon tube
Incubate for 10 min at 37°C incubator on the rocker.
9. Spin down and remove supernatant. Add 100 µl collagenase solution to each tube and incubate for 24 hr at 37°C on rocker.
Make 1 ml in microfuge tube. 1µl CaCl2 stock solution (1 M); 1 µl triton-x-100; 100 µl Tris-HCl pH 7.2 (1 M). Bring to 1 ml with dH2O (898 µl). Dissolve 1 mg collagenase in solution. Make this solution fresh before use. It also works with 0.5 mg collagenase.
10. Spin down worms and remove supernatant. Add 500 µl 1X PBST, incubate on rocker (RT) for 10 min.
11. Spin down worms and remove supernatant. Wash 2X with 500 µl PBST for 5min at RT.
12. Spin down worms and remove supernatant. Incubate for 1 hr in 500 µl AbB buffer (antibody block buffer). Incubate on rocker in the cold room.
13. During this incubation, prepare the antibody solution in AbB.
A place to start:
Use PTL-1 antibody at 1:500 in AbB (so 1µl in 500 µl)
Use Dm1a tubulin antibody at 1:500.
Make the solutions up in the volume needed for all the tubes you plan to test. Keep tubes in dark or wrapped in foil.
14. Spin down worms and remove supernatant. Incubate tubes in 100-500µl volume of particular primary antibody solution. Incubate overnight on rocker in cold room.
15. Spin down worms and remove supernatant. Wash 3X for 5 min each on rocker at RT with 1X PBST. Spin and remove in between each wash.
16. Prepare secondary antibodies in AbB. The ptl-1 antibody was raised in rabbit, so use a goat-anti-rabbit Alexa-fluor 488 at 1:500 (2 µl in 1 ml AbB). The tubulin antibodies were raised in mice, so use a goat-anti-mouse Alexa-fluor 488 at 1:500. Add 200 µl of each secondary antibody to the appropriate tube. Incubate on rocker at RT for 4 hr UNDER FOIL (in dark)
17. Wash 3X 5 min each in 1X PBS
18. Mount by adding 20 µl (or less) Prolong Gold Antifade to drop (10-15 µl) of packed worms. Add coverslip and dry flat overnight IN DARK.
DAY 5- Either examine slides using confocal or move to black box and store in freezer (-20 °C). To examine frozen slides, first thaw box (without opening it) to room temp.